Magnetisable particles are well known. These particles may be used for all affinity separation processes where the targets and non-targets are similar in molecular weight so that techniques such as centrifugation cannot be used. Instead, the targets and non-targets are separated by making use of their different affinity properties. The targets and non-targets can be separated by identifying a ligand such as an antigen, antibody, protein, polysaccharide, etc. which binds to the targets but not to the non-targets. The magnetisable particles are coated with this ligand and added to the sample solution. The solution is then incubated for a period so that as many as possible of the magnetisable particles attach, via the ligand, to the targets. A magnetic field with a gradient is then applied to the solution to attract and thereby trap the magnetisable particles with the targets attached while the remainder of the solution with the non-targets is removed.
The size of the magnetisable particles and the coating applied to them may be varied depending on the application. Typically, the particles may be about 1-10 microns in diameter. In most applications, the magnetisable particles are supplied in a quantity several times the number of targets so that virtually all targets are bound by at least one magnetisable particle.
Various machines have been designed to perform the process of magnetic separation. For example, WO 90/14891 describes a separation device comprising a rack for tubes, with a magnet placed adjacent to each tube slot such that the tubes intersect the fields of force from the magnet. When tubes containing the liquid to be separated are placed in the rack, the magnet attracts the magnetisable particles (and the targets to which they are bound) to the side of the tube. The remaining non-targets can then be removed by a pipette.
When there is a greater quantity of the liquid which is to be separated, the liquid containing the target may be stored within a flexible container or an aseptic bag such as a disposable blood bag. To perform the magnetic separation of the liquid in the bag, the bag is usually laid flat on top of a planar magnet or set of magnets after incubation. The magnetisable particles (with the targets attached) are drawn down towards the magnet. The remaining liquid containing the non-targets can then be evacuated through an outlet tube attached to the bag, while the magnetisable particles and targets are held in place on the bag wall by the magnet. U.S. Pat. No. 4,910,148, WO 90/04019 and WO 91/11716 are all examples of such machines designed for use with cell suspensions.
Separation can be performed either positively or negatively. In positive separation processes, the magnetisable particles attach to the targets which are to be retained so that these desired targets are retained by the magnet. In negative separation processes, the magnetisable particles attach to the non-targets which are to be removed so that these non-targets are retained by the magnet, while the desired targets are withdrawn from the system. In positive separation processes, the desired targets which are retained are still attached to the magnetisable particles, so the magnetisable particles may need to be cleaned from the targets before further analysis. In negative separation processes, the desired targets are withdrawn from the system and are therefore free from magnetisable particles and are ready for further analysis if desired.
U.S. Pat. No. 4,710,472 describes a magnetic separator system where a tube through which the sample flows is held between two magnets or where a magnet is held next to the tube. However, there is not much surface area of contact between the narrow tube and the magnet and therefore separation of the magnetisable particles from the sample is inefficient.
For certain applications of magnetisable particles, such as use in medical treatments, the process and the machines for carrying out the cell isolation process must meet certain requirements set by the relevant authorities, such as the Food and Drug Administration (FDA) in the United States. One such requirement is that the number of magnetisable particles which the system fails to capture must be below a certain threshold. For example, the FDA requirement for medical applications is that there must be less than 100 magnetisable particles per 3 million cells infused into a human subject.
Although a single magnet system can capture most of the magnetisable particles from a cell solution, a proportion of the particles still escape, either through not being captured by the magnet in the first place or through becoming detached from the magnet by turbulence in the flow. Therefore in order to meet the regulatory requirements, systems tend to employ a further magnet downstream of the first. Solution which is evacuated from the area of the first magnet passes into a second area with a second magnet. This second magnet serves to capture the remaining particles which have escaped from or bypassed the first magnet. Systems having two magnetic capture units are generally easily capable of meeting the regulatory requirements, but if stricter requirements are to be met, more magnets downstream of the second magnet may be employed.
Each of the systems mentioned above (U.S. Pat. No. 4,910,148, WO 90/04019 and WO 91/11716) has a primary magnetic capture unit and a secondary magnetic capture unit downstream of the primary unit.
In U.S. Pat. No. 4,910,148 the cell suspension is mixed with the magnetisable particles in a disposable blood bag and incubated. The bag is then placed in the primary magnetic capture unit which comprises a holder above a planar magnet plate in which the bag is held. After the primary separation has taken place, the suspension is transferred to a second blood bag which is attached to the secondary magnetic capture unit which comprises a second planar magnet where the remaining magnetisable particles are trapped, leaving a purified solution.
In WO 90/04019 and WO 91/11716 a similar arrangement is described wherein the cell concentrate is mixed with the magnetisable particles in a first disposable container which is placed in the primary separation chamber with the primary planar magnet. The first container is connected via a tube to a second disposable container which is placed in the secondary separation chamber with the secondary planar magnet. The purified cell concentrate is obtained from the outlet of the second container, while the separated cells attached to the paramagnetic particles are held along the sides of the first and second containers adjacent to the first and second magnets.
In all of these systems, the primary magnetic separation unit tends to have a magnet which has a relatively large field reach, capable of attracting magnetisable particles from the whole width of the first bag. The secondary magnetic separation unit tends to have a magnet which has a smaller field reach, but provides a correspondingly stronger attractive force for retaining the particles. As the fluid is passed through the secondary separation unit, the width of the second bag is restricted so that the magnetisable particles are within the field reach of the second magnet.
A disadvantage of these systems is that the systems require the use of additional disposable containers or bags. For each system run on a two magnet system, at least two bags are used, plus the tubing required to feed the first container, transfer fluid from the first to the second container and extract fluid from the second container. As these bags and tubes must all be sterile and aseptically connected, this represents a significant expense to the end user. These bags must often be the proprietor's own brand bags to fit their particular machine and so may be relatively expensive.
Also the planar magnet assemblies are bulky and heavy, especially when the secondary separation unit is provided with an additional pressing means for restricting the width of the bag. These systems are also complicated and time consuming to set up.